The parasympathetic inhibition of the heart is mediated by the interaction of acetylcholine (ACh) with muscarinic acetylcholine receptors on the muscle surface. This interaction produces a slowing and weakening of the heartbeat. These effects are produced by (a) an increase in a potassium current important in pacemaker activity and (b) a decrease in calcium flux during the action potential. The response of the heart to ACh is interesting partly because the onset and decay of the response are very slow. In this proposed research a combined electrophysiological and biochemical approach is taken to elucidate the molecular and subcellular mechanisms which effectuate the ACh response. (1) The lifetime and elementary conductance of the potassium channels activated by ACh will be determined by noise analysis of agonist induced membrane noise to determine how much of the slowness of the response is due to channel kinetics. (2) The role of membrane protein phosphorylation in mediating the response to ACh will be studied by 32P incorporation and two-dimensional SDS-polyacrylamide gel electrophoresis.